1. Field of the Invention
In general, the present invention is applied to flow cytometry technique. The present invention relates to microspheres, which usually has a diameter of 100 μm or less and is filled with multicolor fluorecent. More specifically, the present invention relates to microspheres that activated esters are held and stabilized on the surface without fluorescence seeping and polymer molecules filled with at least one type of fluorescent dye.
2. Background Art
Polymer particles, filled with fluorescent dye, are often used as a marker or indicator in various biomedical assays. “Microspheres” are minute particles basically having total diameters within micrometer-size. Microspheres can be analyzed with manual techniques or other methods known in the art. Preferably, automation technologies such as flow cytometry disclosed in U.S. Pat. No. 4,665,024 described below, the patent for which was granted to Mansour et al., can be used for analysis of such particles.
Hitherto, biomolecules such as proteins (e.g. antigens and antibodies), nucleic acids, peptides, and sugar chains have been coupled to microparticles filled with at least one color of fluorescent dye. They have been used quantitative analysis of biomolecules or genetic polymorphism analysis. A plurality of analytical items can be simultaneously analyzed by a number of microspheres filled with fluorescent dye of different density and coupled with different types of biomolecules.
The surfaces of microspheres are covered with carboxyl groups. A carboxyl group can form an amide bond with an amino group in a biomolecule via dehydration-condensation reaction. This reaction is used to couple biomolecules on microspheres. However, since the reactivity of carboxyl groups is low, it is necessary to activate the carboxyl groups in some way. There are various types of reagents capable of activating carboxyl groups. Meanwhile, the aforementioned microspheres are filled with at least one fluorescent dye in which fluorescent dye is prone to seep into organic solvent. Thus, the reaction should be carried out in an aqueous solution. EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride) is the only reagent capable of activating carboxyl groups in an aqueous solution. In addition, it is necessary for biomolecules to contain primary amino groups so that they react with activated carboxylic acid esters. In particular, biomolecues such as nucleic acids, sugar chains that have no primary amino groups are necessary to introduce primary amino group into those biomolecule prior to the conjugation.
EDC is a reagent capable of activating carboxyl groups in an acid solvent. The coupling reaction solvent is limited to one used in acidic conditions. However, some types of biomolecules can stably exist only in basic conditions. Thus, the types of biomolecules cannot couple on microspheres. In addition, when nucleic acid couples with microspheres using EDC, EDC may react with amino group of nucleobases under acidic condition.
The following examples pertain to conventional methods for immobilizing biomolecules on fluorescent stained microparticles: a) method for coupling with a biomolecule after activating carboxyl groups carried on the surface of a microsphere to result in succinimide esters; and b) method for coupling with a biomolecule when simultaneously activating carboxyl groups carried on the surface of a microparticle to result in carbodiimide esters.
In the method for activating carboxyl groups to result in succinimide esters described in above a), NHS (N-hydroxysuccinimide) and EDC are added to a suspension of microparticles so as to activate carboxyl groups to result in hydroxysuccinimide esters. The method involves the instantaneous immobilization of a biomolecule on a microparticle, the surface of which has been activated.
In the method for activating carboxyl groups to result in carbodiimide esters described in above b), EDC and biomolecules are simultaneously added to a suspension of microparticles. The method involves the activation of carboxyl groups on the surfaces of microspheres to result in carbodiimide esters such that biomolecules instantaneously react with the active esters, leading to immobilization of the biomolecules on the microspheres.
Active carboxylic acid ester groups such as hydroxysuccinimide ester groups react with hydroxyl groups in an aqueous solution so as to again become carboxyl groups. This reaction is extensively observed, particularly under basic conditions. However, hydroxysuccinimide ester groups are relatively stable in organic solvent without water.
Microspheres are filled with fluorescent dyes in which fluorescent dyes are prone to seep into hydrophobic organic solvents such as aromatic hydrocarbon, pyridine, and dioxane.
JP Patent Publication (Kohyo) Nos. 2001-520323 A and 2002-501184 A described below are known technical literature with regard to methods for producing or using fluorescent stained microparticles.